One-Pot Synthesis of Chiral, Spirocyclic 4-Hydantoin-Proline Derivatives for Incorporation into Spiroligomer-Based Macromolecules.

Derivatives of 4-hydantoin-proline have been synthesized via a direct two-step alkylation method. This method is valuable in the development of applications of N,N'-disubstituted hydantoin bearing α-amino acids by improving yields, reducing the time and number of steps required to synthesize these substituted molecules, and enabling late stage functionalization of spiroligomer termini. Over 20 unique electrophiles have been tested, highlighting the inherent versatility of this chemistry.

A placebo-controlled trial of exenatide twice-daily added to thiazolidinediones alone or in combination with metformin.

AIM: To test the hypothesis that glycaemic control with exenatide added to thiazolidinediones (TZDs) with or without metformin was superior to placebo. METHODS: A 26-week, multi-country (Canada, Mexico, Romania, South Africa and the USA), randomized, double-blind, placebo-controlled study compared exenatide twice-daily vs. placebo in 165 subjects suboptimally controlled with TZDs with or without metformin [HbA(1c) 8.2% (s.d. 0.9), fasting serum glucose 9.1 (2.6) mmol/l, body weight 93.9 (17.8) kg, diabetes duration 6.4 (4.3) years]. After a 2-week, single-blind, lead-in period, subjects were randomly assigned (2 : 1) to add exenatide or placebo to current regimens. The primary endpoint was HbA(1c) change at endpoint (Week 26 or last-observation-carried-forward). RESULTS: Only 8 subjects were treated with concomitant TZD alone. Exenatide reduced HbA(1c) significantly more than placebo [-0.84% (s.e. 0.20) vs. -0.10% (0.23), treatment difference -0.74% (0.16), p < 0.001)]. Mean reductions in body weight were similar in both treatments at endpoint [exenatide, -1.4 (s.e. 0.6) kg vs. placebo, -0.8 (0.7) kg, p = 0.176)]. Nearly 71% of subjects had both a reduction in HbA(1c) and body weight with exenatide compared with 54% with placebo. The most common adverse events (exenatide vs. placebo) were nausea (12% vs. 2%, p = 0.037), vomiting (8% vs. 0%, p = 0.031) and headache (4% vs. 4%). Confirmed (blood glucose <3.0 mmol/l) minor hypoglycaemia was experienced by 4 and 2% of subjects treated with exenatide and placebo, respectively. Incidence of hypoglycaemia was not significantly different between groups. CONCLUSIONS: Exenatide added to TZDs alone or in combination with metformin significantly improved glycaemic control as determined by significant improvement in HbA(1c) without associated hypoglycaemia.

Angiotensin II induces plasminogen activator inhibitor-1 and -2 expression in vascular endothelial and smooth muscle cells.

Angiotensin II (AII)- and Arg8-vasopressin (AVP)-regulated gene expression in vascular cells has been reported to contribute to vascular homeostasis and hypertrophy. In this report, AVP-induced expression of plasminogen activator inhibitor (PAI)-2 mRNA in rat microvessel endothelial (RME) cells was identified using differential mRNA display. Further characterization of vasoactive peptide effects on PAI expression revealed that AII stimulated a 44.8 +/- 25.2-fold and a 12.4 +/- 3.2-fold increase in PAI-2 mRNA in RME cells and rat aortic smooth muscle cells (RASMC), respectively. AII also stimulated a 10- and 48-fold increase in PAI-1 mRNA in RME cells and RASMC, respectively. These AII effects were inhibited by either Sar1, Ile8-angiotensin or the AT1 antagonist DuP 735, but were not significantly altered in the presence of the AT2 antagonist PD123319. AII stimulation of RASMC and RME cells also significantly increased both PAI-1 protein and PAI activity released to the culture medium. Inhibition of protein kinase C completely blocked PMA-stimulated induction of PAI-2 mRNA in both cell types and inhibited the AII-stimulated increase in RASMC by 98.6 +/- 2.8%. In contrast, protein kinase C inhibition only partially decreased the AII-stimulated PAI-2 expression in RME cells by 68.8 +/- 11.1%, suggesting that a protein kinase C-independent mechanism contributes to a 6.9 +/- 1.5-fold AII induction of PAI-2 expression in endothelial cells. AII and PMA also stimulated protein tyrosine phosphorylation in RME cells, and the tyrosine kinase inhibitor genistein partially blocked their induction of PAI-2 mRNA. These findings suggest that AII may regulate plasminogen activation in the vasculature by inducing both PAI-1 and PAI-2 expression.

Temporal expression of c-fos mRNA following balloon injury in the rat common carotid artery.

OBJECTIVE: Restenosis is a common problem which limits the effectiveness of percutaneous transluminal coronary angioplasty (PTCA). The cellular mechanisms of restenosis appear to involve smooth muscle cell (SMC) migration to the neointima in response to mitogens and growth factors, resulting in proliferation and deposition of cells in the lumen of the vessel. An antibody directed against PDGF attenuates this response in the rat. Thus, signaling cascades induced by growth factors including PDGF may be important targets for therapeutic intervention. METHODS: Since a number of growth factors activate c-fos via the p21-ras signaling pathway, we examined c-fos expression in a time course experiment involving restenotic lesions in rat carotid arteries. Sections of arteries collected at 1, 3, 7, 14 and 28 days following balloon injury were hybridized using a fluorescein-labeled RNA probe to c-fos. Immunohistochemistry was performed with antibodies to proliferating cell nuclear antigen (PCNA) and alpha-smc actin to characterize cellular constituents of the neointima, and detect any correlation between fos expression and PCNA localization. RESULTS: Expression of c-fos was low at day 1. By day 3, the media and adventitia were positively stained. At days 7 and 14, most cells in the neointima were labeled. By day 28, c-fos was expressed mainly in scattered cells along the luminal surface. Control sections revealed little labeling and confirmed specific staining by the antisense strand, PCNA localization and c-fos expression were similar at days 1, 3, 7 and 28, but at day 14 c-fos was expressed throughout the lesion, with PCNA localized mainly along the luminal edge. The majority of the cells making up the neointima stained rather intensely for alpha-smc actin, identifying them as SMCs. CONCLUSIONS: Results of these experiments indicate that, while c-fos expression correlates with lesion formation, it may be associated with a cellular process distinct from proliferation in this model.

Isolation of mycobacteria from indoor swimming pools in Finland.

The presence of mycobacteria in seven indoor pools in Finland was evaluated by multiple culture methods. Replicate samples, with and without inactivation of chlorine by sodium thiosulfate, were cultured in two laboratories. Laboratory I used two methods: (A) no decontamination and (B) cetylpyridinium chloride (0.005%, 20 min); and Laboratory II two methods: (C) cetylpyridinium chloride (0.005%, 18 h) and (D) oxalic acid (5%, 15 min). Samples processed by methods (A) and (B) were cultured on different egg media of pH 6.3 or 5.8; by method (C) on Middlebrook and Cohn 7H10 (+OADC) agar of pH 5.5; and by method (D) on Middlebrook and Cohn 7H10 agar (+OADC) with cycloheximide (500 microg/ml). Mycobacteria were recovered from five (71%) of seven pools. Detection of mycobacteria depended on the method used. High isolation rates (36-46% of the samples) were obtained by methods (A), (B) and (D). Contamination was a problem only with method (A). Inactivation of chlorine had a variable impact on mycobacterial detection. Isolates included M. kansasii, M. gordonae, M. fortuitum complex, M. sphagni, and M. vaccae, as well as M. simiae-like and M. chubuense-like organisms. In addition, a group of slowly growing and a group of rapidly growing isolates with previously unknown fatty acid and alcohol composition were isolated. No M. avium was detected. Mycobacterial counts were highest in a small pool with high temperature, low pH, and low content of free available chlorine.

NF-kappa B is activated during acute inflammation in vivo in association with elevated endothelial cell adhesion molecule gene expression and leukocyte recruitment.

Leukocytes accumulate at sites of inflammation in response to the induced expression of endothelial cell adhesion molecules. The nuclear transcription factor kappa B (NF-kappa B) plays a critical role in the cytokine-induced expression of these genes in cultured endothelium. We examined the relationship between NF-kappa B activation and endothelial cell adhesion molecule gene expression in vivo during the initiation of acute inflammation. Nuclear NF-kappa B DNA-binding activity was rapidly increased within lung and heart tissues of rats administered endotoxin, consistent with the translocation of NF-kappa B complexes from the cytoplasm to the nucleus. This NF-kappa B was composed of p50 and p65 subunits, and could bind NF-kappa B elements in the E-selectin promoter. NF-kappa B activation was maximal within 30 min and persisted for at least 3 hr after endotoxin treatment. NF-kappa B activation preceded the transcriptional activation of the P-selectin, E-selectin, VCAM-1, and ICAM-1 genes. In the lung, increased expression of P-selectin and ICAM-1 protein was detected immunohistochemically. These molecular events were temporally associated with the sequestration of leukocytes and the development of pulmonary inflammation. NF-kappa B activation is therefore an early event in the initiation of acute inflammation in vivo. This molecular pathway may be of consequence in the pathogenesis of acute inflammatory disease.

Hypoxic regulation of vascular endothelial growth factor in retinal cells.

BACKGROUND: Vascular endothelial growth factor (VEGF) is an angiogenic protein and vasopermeability factor whose intraocular concentrations are closely correlated with active neovascularization in patients with diabetes mellitus, central retinal vein occlusion, retinopathy of prematurity, and rubeosis iridis. OBJECTIVE: To determine whether hypoxia could induce expression of VEGF in retinal cells, which then promotes retinal endothelial cell proliferation. METHODS: Retinal pigment epithelial cells, pericytes, and microvascular endothelial cells were exposed to hypoxic conditions in vitro, and RNA expression of VEGF was evaluated by Northern blot analysis. The VEGF-specific proliferative potential of the medium was measured by means of retinal endothelial cell growth assays and VEGF-neutralizing VEGF receptor IgG chimeric protein. RESULTS: The VEGF RNA levels increased within 4 hours and reached elevations of threefold to 30-fold after 18 hours of hypoxia (0% to 5% oxygen, 5% carbon dioxide, 90% to 95% nitrogen) in all cell types (.01 < P < .03). Stimulation was dependent on oxygen concentration. The VEGF RNA levels were normalized by reinstitution of normoxia for 24 hours (P < .004). Medium conditioned by hypoxic retinal pericytes and retinal pigment epithelial cells stimulated retinal endothelial cell growth by 20% (P = .04), and this stimulation was entirely inhibited by VEGF-neutralizing receptor chimeric protein (P = .02). CONCLUSION: Hypoxia increases VEGF expression in retinal cells, which promotes retinal endothelial cell proliferation, suggesting that VEGF plays a major role in mediating intraocular neovascularization resulting from ischemic retinal diseases.

Genetics of familial neuroblastoma: long-range studies.

This is a longitudinal study of a family prone to neuroblastoma. The family was originally reported in 1975 when three children in a single generation were found to be affected. We now report the case of a fourth affected child, the sole child born in the succeeding generation. Cytogenetic studies have disclosed the segregation in the family of a paracentric inversion of the long (q) arm of chromosome No. 11 and a deletion of the short (p) arm of chromosome No. 21. However, the independent assortment of the inv(11q) and 21p- chromosomes with neuroblastoma permits us to exclude them as linkage markers for the neuroblastoma gene.

Leukemia inhibitory factor and ciliary neurotrophic factor regulate dendritic growth in cultures of rat sympathetic neurons.

Cytokines such as leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) have previously been shown to regulate neurotransmitter and neuropeptide synthesis in sympathetic neurons [P.H. Patterson, Leukemia inhibitory factor, a cytokine at the interface between neurobiology and immunology, Proc. Natl. Acad. Sci. USA 91 (1994) 7833-7835]. We considered the possibility that these agents may also affect the development of neuronal cell shape. Intracellular dye injection and immunocytochemistry were used to assess dendritic growth in cultures of perinatal rat sympathetic neurons and the effects of LIF and CNTF were compared to those of osteogenic protein-1 (OP-1), a growth factor that induces profuse dendritic growth in these neurons [P. Lein, M. Johnson, X. Guo, D. Rueger, D. Higgins, Osteogenic protein-1 induces dendritic growth in rat sympathetic neurons, Neuron 15 (1995) 597-605]. Under control conditions, sympathetic neurons formed only axons. Exposure to either LIF or OP-1 stimulated dendritic growth, but the magnitude of the response to LIF was much less than that obtained with OP-1 with respect to both dendritic number and length. Simultaneous exposure to LIF and OP-1 resulted in dendritic growth equivalent to that observed in the presence of LIF alone, suggesting that LIF inhibits the response of neurons to OP-1. Both the stimulatory and inhibitory effects of LIF were mimicked by CNTF, but not by other growth factors. These data suggest that LIF and CNTF regulate dendritic development in a complex manner that is dependent on both the morphological state of the neuron and the presence of other growth factors. However, the net effect of exposure to these cytokines appears to be the production of a population of neurons with rudimentary arbors consisting of only one or two short dendrites.

A comparison of twice-daily exenatide and biphasic insulin aspart in patients with type 2 diabetes who were suboptimally controlled with sulfonylurea and metformin: a non-inferiority study.

AIMS/HYPOTHESIS: The aim of this 52-week, open-label, non-inferiority trial was to compare the safety and efficacy of exenatide (an incretin mimetic) with that of biphasic insulin aspart. MATERIALS AND METHODS: Patients on metformin and a sulfonylurea were randomised to exenatide (n = 253; 5 microg twice daily for 4 weeks, 10 microg thereafter) or biphasic insulin aspart (n = 248; twice-daily doses titrated for optimal glucose control), while continuing with metformin and sulfonylurea treatment. RESULTS: Glycaemic control achieved with exenatide was non-inferior to that achieved with biphasic insulin aspart (mean+/-SEM, HbA(1c) change: exenatide -1.04 +/- 0.07%, biphasic insulin aspart -0.89 +/- 0.06%; difference -0.15 [95% CI -0.32 to 0.01]%). Exenatide-treated patients lost weight, while patients treated with biphasic insulin aspart gained weight [between-group difference -5.4 (95% CI -5.9 to -5.0) kg]. Both treatments reduced fasting serum glucose (exenatide -1.8 +/- 0.2 mmol/l, p < 0.001; biphasic insulin aspart -1.7 +/- 0.2 mmol/l, p < 0.001). Greater reductions in postprandial glucose excursions following morning (p < 0.001), midday (p = 0.002) and evening meals (p < 0.001) were observed with exenatide. The withdrawal rate was 21.3% (54/253) for exenatide and 10.1% (25/248) for biphasic insulin aspart. Nausea (33% incidence, 3.5% discontinuation) was the most common adverse event observed with exenatide. CONCLUSIONS/INTERPRETATION: Exenatide treatment resulted in HbA(1c) reduction similar to biphasic insulin aspart and provided better postprandial glycaemic control, making it a potential alternative for the treatment of type 2 diabetes. Treatment with biphasic insulin aspart was associated with weight gain and lower risk of adverse gastrointestinal events. Although the availability of glucose-lowering agents associated with weight reduction may be considered a therapeutic advance, the long-term implications of progressive weight reduction observed with exenatide have yet to be defined.

Entropy-driven stabilization of a novel configuration for acceptor-hydrogen complexes in GaN.

We present a model for the microscopic structure of Mg-H complexes in GaN, explaining the unusual bond angle observed in recent vibrational spectroscopy studies. The structure is not the lowest-energy configuration at T = 0, but it is stabilized at elevated temperatures due to the large entropy associated with a set of low-energy rotational excitations. The rotational excitation spectrum is calculated using a quantum-mechanical model in which the hydrogen atom moves in a weak corrugation potential. Consequences for experiment are discussed.